沙门菌是人类一种常见的食源性致病菌,沙门菌主要污染肉类、鱼、禽、奶、蛋等食品.食用了这些未煮透的污染食品是引起沙门菌食物中毒的最主要原因.研制能对食品中的沙门菌进行现场快速精准检测的试剂盒,对于保障人们的食品安全具有重要意义.本研究以沙门菌的fimY基因作为特异性的检测靶基因,建立一种可视化、低成本的环介导等温扩增技术,以快速检测食源性沙门菌.建立的LAMP方法具有良好的特异性,对肠出血性大肠杆菌O157、肠毒性大肠杆菌ETEC、空肠弯曲菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌、单增李斯特菌等其他食源性致病菌和空白对照(水)的检测结果均为阴性.灵敏性检测结果表明,建立的LAMP检测沙门菌的灵敏度为3.8×101 CFU/mL,而常规PCR检测沙门菌的灵敏度为3.8×104 CFU/mL,LAMP法检测比常规PCR检测的灵敏度高1000倍.另外,采用建立的LAMP方法对18株鸡源和猪源的沙门菌分离株进行检测,结果均为阳性,与基于invA基因的常规PCR结果的一致.本研究中建立的LAMP检测方法为食源性沙门菌的检测提供一种可视化、灵敏、成本低的快速检测技术.

单核细胞增生李斯特菌(Lm)是主要的食源性致病菌之一,也是一种重要的人畜共患病原菌,感染人和动物后主要表现为败血症、脑膜炎和单核细胞增多症等.为建立快速检测Lm的方法,本研究以Lm毒力因子actA作为特异性检测的靶基因,通过条件优化初步建立了快速检测食源性Lm的环介导等温扩增(LAMP)方法.并采用该方法进行了特异性、敏感性及临床样品检测试验.结果显示,利用建立的LAMP方法检测Lm、肠出血性大肠杆菌O157∶H7、鼠伤寒沙门氏菌、金黄色葡萄球菌、小肠结肠炎耶尔森菌、产气荚膜梭菌、空肠弯曲菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、多杀性巴氏杆菌、猪链球菌、猪丹毒丝菌或支气管败血波氏杆菌基因组DNA,除 Lm为阳性外,其他病原菌均为阴性,表明该方法的特异性较强;将Lm菌液10倍倍比稀释为4.7×109cfu/mL～4.7×100cfu/mL,再采用该LAMP方法和常规PCR方法进行检测,结果显示,LAMP对Lm的检测下限为4.7×101cfu/mL,而常规PCR对Lm的检测下限为4.7×103cfu/mL,即LAMP比普通PCR的敏感性高100倍,表明本研究建立的LAMP方法敏感性较高;采用建立的LAMP方法对118份猪肉临床样品进行检测,结果显示有3份样品为阳性,与常规PCR和国标的检测结果均一致.本研究基于Lm actA基因建立了Lm LAMP检测方法,且该方法具有特异性强、敏感性高、结果可视等优点,适用于实验室和基层食品安全监管部门的现场快速检测.

通过建立雏鹅高尿酸血症模型,研究高蛋白日粮对雏鹅血清尿酸水平、肝脏和肾脏超微结构及AB-CG2表达的影响.超微结构观察发现,对照组(CP)肝脏和肾脏细胞器结构完好,高蛋白1组(HP1)、高蛋白2组(HP2)肝脏部分线粒体开始出现肿胀、空泡变性,内质网肿胀消失,糖原减少.肾脏细胞部分线粒体开始出现肿胀、空泡变性,核膜间隙增大.雏鹅7日龄时,HP2组鹅的血清尿酸水平极显著高于CP组和HP1组(P<0.01);雏鹅14日龄时,HP2组鹅的血清尿酸水平极显著高于CP组和HP1组(P<0.01),HP1组鹅血清尿酸水平极显著高于CP组(P<0.01).qPCR、Western-boltting及免疫组化结果显示,HP2组鹅十二指肠、空肠中ABCG2基因和蛋白的表达显著或极显著高于CP组和HP1组(P<0.05或P<0.01),鹅肾脏ABCG2基因和蛋白的表达显著或极显著低于CP组和HP1组(P<0.05或P<0.01);HP1组鹅ABCG2基因和蛋白在空肠中的表达显著高于CP组(P<0.05),在肾脏中的表达显著低于CP组(P<0.05).综上所述,饲喂高蛋白日粮导致雏鹅肝脏、肾脏细胞受损,血清尿酸水平升高,引发雏鹅高尿酸血症,空肠、十二指肠中ABCG2的表达增加,而肾脏中ABCG2的表达降低.

[目的]探讨高蛋白日粮对雏鹅生长性能、血清炎症因子及肝脏、肾脏、空肠、十二指肠中黄嘌呤脱氢酶(XDH)表达的影响.[方法]选用1日龄雁鹅72只,随机分成对照组(CP)、高蛋白组1(HP1)和高蛋白组2(HP2),分别饲喂含180.00,230.00和280.00 g/kg粗蛋白的日粮,每组3个重复,每个重复8只鹅.试验为期14 d,每周测定各组雏鹅采食量、体质量.于7和14 d采血,分离血清,用ELISA法测定血清中黄嘌呤氧化酶(XOD)活性及肿瘤坏死因子(TNF-α)、白细胞介素1β(1L-1β)、白细胞介素6(IL-6)、尿酸(UA)含量;采用RT-qPCR、免疫组化法、West-ern-blotting法测定14日龄雏鹅肝脏、肾脏、空肠、十二指肠中XDH基因及其蛋白的表达水平.[结果])7日龄时,HP2组雏鹅的平均体质量显著小于CP组(P＜0.05),极显著小于HP1组(P＜0.01),HP1组雏鹅的平均日增重显著大于HP2组(P＜0.05);14日龄时,CP组雏鹅的平均体质量、平均日增重、日采食量极显著高于HP1和HP2组(P＜0.01).2)7日龄时,HP2组雏鹅血清中的1L-1β、TNF-α、UA含量及XOD活性均极显著高于CP组(P＜0.01),1L-1β、TNF-α、UA含量显著高于HP1组(P＜0.05);HP1组雏鹅血清中TNF-α质量浓度极显著高于CP组(P＜0.01).14日龄时,HP2组雏鹅血清中1L-1β、IL-6、TNF-α、 UA含量及XOD活性均显著或极显著高于CP组和HP1组;HP1组雏鹅血清中的1L-1β、IL-6、TNF-α、UA含量及XOD活性极显著高于CP组(P＜0.01).3)HP2组雏鹅肝脏、肾脏、十二指肠的XDH基因及其蛋白的表达显著或极显著高于CP组(P＜0.05或P＜0.01),肝脏、肾脏中XDH基因及其蛋白的表达显著或极显著高于HP1组(P＜0.05或P＜0.01);HP1组雏鹅肝脏、十二指肠中XDH基因及其蛋白的表达显著或极显著高于CP组(P＜0.05或P＜0.01).各组雏鹅空肠中XDH基因及其蛋白的表达无显著差异(P＞0.05).[结论]摄入过高水平的蛋白会导致雏鹅生长性能降低,机体发生炎症反应,肝脏、肾脏、十二指肠中XDH基因和蛋白表达量增加,从而引发雏鹅高尿酸血症.

[目的]采用色谱-质谱联用的代谢组学方法分析高蛋白日粮对雏鹅血清中内源性小分子代谢产物的影响.[方法]选用1日龄雁鹅48只,随机分成2组,每组24只鹅.对照组(CP)饲喂基础日粮(含18％粗蛋白),高蛋白组(HP)饲喂含23％粗蛋白的日粮,试验期为14 d.[结果]:与对照组相比,雏鹅7、14日龄时,高蛋白组鹅血清中谷丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、乳酸脱氢酶活性和总蛋白、尿酸含量极显著升高(P<0.01),球蛋白含量显著升高(P<0.05);14日龄时,高蛋白组鹅血清中总胆红素、间接胆红素、尿素、肌酐含量极显著升高(P<0.01).正交偏最小二乘-判别分析法((OPLS-DA))分析结果显示,2组间有明显的分离趋势,提示HP组与CP组血清代谢谱有差异.对LC-TOF/MS和GC-TOF/MS分析结果进行代谢富集和途径分析表明,氨酰tRNA生物合成、嘌呤代谢、嘧啶代谢、β-丙氨酸代谢、赖氨酸降解、磷酸肌醇代谢、泛酸和辅酶A生物合成途径发生了显著改变.热图分析显示高蛋白组和对照组差异代谢物含量显著不同.[结论]本试验通过非靶向代谢组学筛选出的雏鹅血清中黄嘌呤、瓜氨酸、尿酸等41种代谢物与高蛋白日粮诱导雏鹅高尿酸血症发生相关,为潜在标志物.

Riemerella anatipestifer causes epizootic infectious disease in poultry resulting in serious economic losses especially to the duck industry. In our previous study, R. anatipestifer was found to lyse duck erythrocytes in vitro. In the present study, a random Tn4351 mutagenesis library of hemolytic R. anatipestifer strain SX containing 4000 mutants was constructed to investigate the genetic basis of hemolytic activity. Thirty mutants with reduced hemolytic activity and one with increased hemolytic activity were screened and insertions in 24 genes were identified. Of these genes, four were predicted to encode outer membrane proteins, one encoded a cytoplasmic membrane protein, 11 encoded cytoplasmic proteins, and eight encoded proteins with unknown locations. Based on current annotations of the R. anatipestifer genomes, of the 24 genes, 7 (29.17%) were involved in iron utilization. The hemolytic activities of the complemented strains M2 (pRES-Riean_0790) and M18 (pRES-Riean_0653) were restored, indicating that both Riean_0653 and Riean_0790 are involved in the hemolytic activity of strain SX. However, the recombinant proteins rRiean_0317, rRiean_0790, rRiean_0653, rRiean_1027, rRiean_1143, and rRiean_1561 had no hemolytic activity, suggesting that none were hemolysins.

[目的]旨在研究饲喂高钙、高蛋白日粮对肉仔鸡生长性能、血清生化指标、抗氧化指标及盲肠菌群的影响.[方法]选取160只体质量相近的健康1日龄肉仔鸡,随机分为4组,每组5个重复,每个重复8只,分别为对照组(CP含量21％,Ca含量1％)、高蛋白组(CP含量31％,Ca含量1％)、高钙组(CP含量21％,Ca含量5％)和高钙高蛋白组(CP含量31％,Ca含量5％).[结果]结果表明:(1)仔鸡21日龄时,对照组鸡的平均体质量、日增质量、日采食量均极显著高于其他3组(P<0.01),料重比极显著小于高钙高蛋白组(P<0.01);高蛋白组鸡的平均体质量、日采食量显著高于高钙组和高钙高蛋白组(P<0.05);高钙组鸡的平均体质量、日增质量显著高于高钙高蛋白组(P<0.05)而料重比显著小于高钙高蛋白组(P<0.05).(2)高钙、高蛋白日粮可导致仔鸡血清中AST、ALT、UA、CREA-S、UREA、GSH-Px、SOD、MDA、T-AOC、XOD活性或含量异常.(3)IlluminaMiseq高通量测序结果表明,仔鸡盲肠中的厚壁菌门Firmicutes、变形菌门Proteobacteria、厌氧杆菌属Barnesiella、另枝菌属Alistipes、粪便杆菌属Faecalibacterium、理研菌属Rikenella、卟啉单胞菌属Porphyromonas、梭状芽孢杆菌属第十八类群Clostridium_ⅩⅧ、嗜碱菌Alkaliphilus、颤杆菌克属Oscillibacter、梭状芽孢杆菌Clostridium的物种丰富度发生显著变化(P<0.01或P<0.05);PICRUSt功能预测结果显示,葡萄糖和一些氨基酸和核苷酸的合成代谢出现紊乱.(4)对照组鸡肝肾组织正常,结构清晰;其他三组鸡肝肾组织发生不同程度的病变.[结论]高钙高蛋白日粮导致仔鸡生长性能、抗氧化能力降低,肝肾功能受损,引起仔鸡高尿酸血症及盲肠菌群结构异常.

Riemerella anatipestifer causes epizootic infectious disease in ducks, geese, turkeys and other birds, and serious economic losses especially to the duck industry. However, little is known about the molecular basis of its pathogenesis. In this study, signature-tagged transposon mutagenesis based on Tn4351 was developed in R. anatipestifer to identify genes essential for survival and pathogenesis. Seventeen tagged Tn4351 random mutation libraries of the R. anatipestifer strain WJ4 containing 5100 mutants were screened for survive using a duckling infection model. Twenty mutants that could not be recovered from the infected ducklings, were identified, and 17 mutated genes were identified by inverse PCR or genome-walking PCR. Of these genes, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains, and FIP52_03215 and FIP52_03175 were found exclusively on the chromosome of serotype 1 R. anatipestifer strains. Twelve out of 17 genes encoding for proteins were predicted to be involved in amino acid, nucleotide, coenzyme, or lipid transport and metabolism, one gene was predicted to be involved in signal transduction, one gene was predicted to be involved in DNA replication, recombination and repair, the other three genes had an unknown function. Animal experiments showed that the virulence of mutants 16-284, 7-295, 24-231, 9-232 and 19-214 were significantly attenuated compared to that of the wild-type WJ4. Moreover, the median lethal dose of mutant 16-284 was greater than 10(10) CFU, and its virulence to ducklings was partially restored when it was complemented with the shuttle expression plasmid pRES-FIP52_09345. The results in this study will be helpful to further study the molecular mechanisms of the pathogenesis of R. anatipestifer infection.